Summary

Embryonic stem (ES) cells can be derived and propagated from multiple
strains of mouse and rat through application of small-molecule inhibitors of
the fibroblast growth factor (FGF)/Erk pathway and of glycogen synthase kinase
3. These conditions shield pluripotent cells from differentiation-inducing
stimuli. We investigate the effect of these inhibitors on the development of
pluripotent epiblast in intact pre-implantation embryos. We find that blockade
of Erk signalling from the 8-cell stage does not impede blastocyst formation
but suppresses development of the hypoblast. The size of the inner cell mass
(ICM) compartment is not reduced, however. Throughout the ICM, the
epiblast-specific marker Nanog is expressed, and in XX embryos epigenetic
silencing of the paternal X chromosome is erased. Epiblast identity and
pluripotency were confirmed by contribution to chimaeras with germline
transmission. These observations indicate that segregation of hypoblast from
the bipotent ICM is dependent on FGF/Erk signalling and that in the absence of
this signal, the entire ICM can acquire pluripotency. Furthermore, the
epiblast does not require paracrine support from the hypoblast. Thus,
naïve epiblast and ES cells are in a similar ground state, with an
autonomous capacity for survival and replication, and high vulnerability to
Erk signalling. We probed directly the relationship between naïve
epiblast and ES cells. Dissociated ICM cells from freshly harvested late
blastocysts gave rise to up to 12 ES cell clones per embryo when plated in the
presence of inhibitors. We propose that ES cells are not a tissue culture
creation, but are essentially identical to pre-implantation epiblast
cells.

INTRODUCTION

During early murine development, successive differentiation events lead to
the segregation of firstly the trophectoderm, which forms an outer epithelial
layer, and secondly the hypoblast on the blastocoelic surface of the inner
cell mass (ICM). The remaining internal cells form the epiblast from which the
foetus will arise. Subsequent to implantation in the uterus, the epiblast
forms an epithelial structure that undergoes rapid cell division. Embryonic
stem (ES) cells can be derived from the preimplantation epiblast
(Batlle-Morera et al., 2008;
Brook and Gardner, 1997;
Evans and Kaufman, 1981;
Martin, 1981), but have not
been derived from the post-implantation epiblast
(Brons et al., 2007;
Tesar et al., 2007). The
relationship between pluripotent cells that exist transiently in the mammalian
embryo and immortal ES cells in culture has not been fully defined
(Gardner and Brook, 1997;
Smith, 2001). On the one hand,
ES cells have only been authenticated for a few mouse strains. This has been
used to argue that genetic and/or epigenetic factors determine their
derivation, and that they may be considered an artefact of cell culture
(Buehr and Smith, 2003). On the
other hand, the ability of ES cells to re-enter embryonic development and
contribute extensively to chimaeras indicates that any adaptations to culture
are fully and rapidly reversible (Bradley
et al., 1984).

The dilemma surrounding definition of the status of ES cells has remained
unresolved largely because the process of ES cell derivation from embryos has
historically been inefficient and reliant on poorly defined and variable
culture conditions and protocols. However, an understanding of the
requirements for propagating ES cells has evolved progressively since the
initial reports in which a feeder layer of mitotically inactivated fibroblasts
was used in combination with culture medium containing foetal calf serum
(Evans and Kaufman, 1981;
Martin, 1981). The feeder
layer was then substituted with the cytokine leukaemia inhibitory factor (LIF)
(Smith et al., 1988;
Williams et al., 1988) and,
subsequently, serum was replaced with bone morphogenetic protein 4
(Ying et al., 2003). Most
recently, defined conditions free from feeder cells, serum and cytokines were
established based on combinations of small-molecule inhibitors of the
fibroblast growth factor (FGF)/mitogen-activated protein kinase
(Mek)/extracellular signal-related kinase (Erk1/2; Mapk3/1 - Mouse Genome
Informatics) pathway and of glycogen synthase kinase 3 (Gsk3)
(Ying et al., 2008). These
defined conditions, known as 3i or 2i, have been applied to the derivation of
ES cells and have enabled establishment of authentic ES cells from hitherto
recalcitrant strains, such as non-obese diabetic (NOD) mice
(Nichols et al., 2009).
Furthermore, they have been applied to rat embryos, resulting in the
production of the first germline-competent rat ES cells
(Buehr et al., 2008;
Li et al., 2008). The use of
3i/2i for efficient derivation of ES cells provoked the hypothesis that
immortalising the pluripotent cells from developing embryos does not depend
upon adaptation and selection in culture, but rather that the epiblast might
be in a ground state that can be maintained by blocking inductive
differentiation pathways. Thus, we speculated that cells with properties
identical to ES cells exist, at least transiently, in the epiblast of the
preimplantation embryo.

The roles of the extra-embryonic lineages, trophectoderm and hypoblast, in
the development of a functional epiblast have not been described.
Specification and maintenance of the trophectoderm in murine embryos are
dependent upon activation of a sequence of transcription factors, including
Tead4, Cdx2 and eomesodermin (Eomes)
(Nishioka et al., 2009;
Strumpf et al., 2005;
Voss et al., 2000;
Yagi et al., 2007). FGF/Erk
signalling has been implicated in the differentiation of trophoblast
(Lu et al., 2008) and in the
proliferation of diploid trophoblast (Arman
et al., 1998; Nichols et al.,
1998; Tanaka et al.,
1998). There have been reports implicating Erk signalling in the
formation and propagation of the hypoblast. Embryos lacking Grb2, a key
adaptor of the receptor tyrosine kinase signalling pathway, are defective in
the formation of the hypoblast (Cheng et
al., 1998). In the early embryo, Fgf4 is a potent activator of Erk
signalling, and has been suggested to play a role in the maintenance of
hypoblast (Silva, J. M. et al.,
2008). Furthermore, addition of Fgf4 to isolated ICMs in culture
promotes outgrowth of parietal endoderm, a derivative of the hypoblast
(Rappolee et al., 1994).
Reducing Erk activity has been shown to enhance the efficiency of ES cell
derivation by promoting retention of Oct4 (Pou5f1)-positive epiblast during
the outgrowth phase (Buehr and Smith,
2003). Also, the differentiation of established ES cells and their
requirement for LIF can be diminished by addition of synthetic inhibitors of
Erk signalling (Burdon et al.,
1999).

Here, we examine the relationship and identity between pluripotent
naïve epiblast and cultured ES cells. We investigate the effect of 3i/2i
and the component inhibitors on the development of the embryonic and
extra-embryonic lineages in preimplantation mouse embryos.

MATERIALS AND METHODS

Mice and embryos

The strains of mice used in this study were 129/Sv, C57BL/6/Ola, CBA/Ca,
and F1 hybrids between C57BL/6/Ola and CBA/Ca. They were maintained by
in-house breeding on a lighting regime of 14 hours light and 10 hours darkness
with food and water supplied ad libitum. Prior to caging with stud males,
females were selected for oestrous by visual inspection of the vagina
(Champlin et al., 1973).
Detection of a copulation plug the following morning was used to confirm
successful mating; the resulting embryos were then considered to be 0.5 days
post-coitum (dpc). For all experiments, the embryos from at least two females
were pooled and randomly assigned to experimental groups.

Embryo culture

Zygotes in cumulus masses were dissected from oviduct ampullae at 0.5 dpc
and placed in hyaluronidase (300 μg/ml, Sigma, Gillingham, UK) in M2 medium
(Quinn et al., 1982) (made in
house), to disperse the cumulus cells before rinsing in M2 medium. Embryos at
the 8-cell stage were flushed from oviducts at 2.5 dpc; blastocysts were
flushed from uteri at 3.5 or 3.75 dpc using M2. Embryos from the 1-cell stage
to blastocyst were cultured in KSOM (MR-020P-5D, Millipore, Watford, UK). From
the expanded blastocyst stage, embryos were cultured in N2B27 medium
(Nichols and Ying, 2006;
Ying and Smith, 2003). Where
appropriate, inhibitors were added at the following concentrations and in the
combinations specified in the text: PD184352, 0.8 μM; PD0325901, 1 μM;
Chir99021, 3 μM (all synthesized in the Division of Signal Transduction
Therapy, University of Dundee, Dundee, UK); SU5402 (Calbiochem, San Diego, CA,
USA), 2 μM; PD173074 (Sigma), 100 nM.

Confocal analysis

Confocal images were collected using a Leica TCS SP5 confocal microscope.
Reconstructions of three-dimensional images from confocal sections and cell
counts were performed using Leica software and Adobe Photoshop.

Statistical analysis

Probability (P) values were established using Student's
t-test for comparison between two samples.

Clonal analysis of ES cells from ICMs

Embryos at the 8-cell stage were flushed from oviducts of 129/Sv mice at
2.5 dpc, cultured in KSOM+2i for 2 days, then transferred to
N2B27+2i±LIF for one more day. Alternatively, peri-implantation embryos
were flushed directly from uteri of F1 mice that had been mated with F1 males
4.5 days previously. Embryos were subjected to immunosurgery to remove the
trophectoderm using the protocol described previously
(Nichols et al., 1998;
Solter and Knowles, 1975).
Isolated epiblasts were then disaggregated using trypsin. Residual clumps and
obviously dying cells were discarded and the resulting single cells or
couplets were deposited individually into each well of a gelatinised 96-well
plate containing N2B27+2i±LIF. After 8 days, colonies were fixed and
scored by morphology or immunostaining. In some cases, colonies were passaged
further and injected into blastocysts for chimaera formation.

Chimaera analysis

Ten to 12 dissociated cells from immunosurgically isolated ICMs or ES cell
cultures were injected into C57BL/6/Ola blastocysts. Injected embryos were
transferred to the uteri of C57BL/6/Ola × CBA/Ca F1 females, previously
mated with vasectomised males, at 2.5 dpc. Chimaeras were identified by coat
colour mosaicism and tested for germline transmission by mating with an
appropriate wild-type mouse.

RESULTS

Inhibition of Erk activity in early embryos eliminates hypoblast and
expands epiblast

We previously demonstrated that ES cells can be maintained in an
undifferentiated state in the presence of the Mek inhibitor PD184352 and the
FGF receptor inhibitor SU5402, but that survival and expansion are enhanced by
addition of an inhibitor of Gsk3, Chir99021
(Ying et al., 2008). We
therefore cultured mouse embryos from the 8-cell stage in medium supplemented
with PD184352 and SU5402, with and without Chir99021. In all conditions,
expanded blastocysts formed and many of these hatched from the zona pellucida,
indicating that the inhibitors do not interfere with trophoblast formation and
differentiation. We then examined the ICMs by confocal immunofluorescence
analysis. We utilised specific antibodies raised against Oct4 to identify
cells of the epiblast and early hypoblast
(Palmieri et al., 1994), and
against Nanog, which is specific for the epiblast
(Chambers et al., 2003;
Mitsui et al., 2003), and
Gata4, a hypoblast marker (Morrisey et
al., 1996). In control cultured embryos, Gata4-positive cells were
relatively abundant and Nanog-positive cells were scarce
(Fig. 1A,B). By contrast,
embryos cultured in PD184352 and SU5402 showed a dramatic reduction in
Gata4-positive cells (Fig.
1A,B; P<0.0001). They also exhibited a slight increase
in the number of cells in the Nanog-positive compartment, and this was greatly
enhanced by the addition of Chir99021 (3i,
Fig. 1A,B; P=0.003 and
P<0.0001 for PD184352+SU5402 versus control and 3i versus control,
respectively). Embryos cultured in Chir99021 alone did not differ from
untreated controls (Fig.
1A,B).

We substituted both PD184352 and SU5402 with the more potent Mek inhibitor
PD0325901, or with PD173074, which has a higher affinity than PD184352 for the
FGF receptor and is more selective than SU5402
(Bain et al., 2007). In the
presence or absence of Chir99021, there were few Gata4-positive cells compared
with controls, similar to the embryos cultured in 3i
(Fig. 1C,D;
P<0.0001). Interestingly, the Nanog-positive compartment was
expanded in both cases without addition of Chir99021
(Fig. 1C,D;
P<0.0001 comparing either condition with controls, and
P=0.88 and P=0.21 for PD0325901 and PD173074, respectively,
compared with 2i). This suggests that off-target effects of SU5402 might
inhibit ICM expansion in a manner that can be rescued by Gsk3 inhibition. The
combination of PD0325901 with Chir99021, termed 2i, is very effective for
propagating ES and induced pluripotent cells
(Buehr et al., 2008;
Silva, J. et al., 2008;
Ying et al., 2008). For
short-term culture of 2 days, 8-cell stage embryos cultured in PD0325901 or
PD173074 alone were virtually indistinguishable from those cultured in 2i
(Fig. 1C,D).

Effect of FGF/Erk and Gsk3 inhibition on inner cell mass
development. (A) Confocal images of mouse embryos grown from the
8-cell stage (E2.5) for 3 days in control medium, in 3i (medium supplemented
with Chir99021, PD184352 and SU5402), medium supplemented with Chir99021
alone, or medium supplemented with PD184352 and SU5402. Embryos were
immunostained using antibodies raised against Nanog (green) and Gata4 (blue).
(B) Bar chart showing cell numbers of epiblast (Nanog positive, green)
and hypoblast (Gata4 positive, blue) of embryos cultured in the conditions
shown in A. Bars indicate the mean ± s.d. (C) Confocal images of
embryos grown from the 8-cell stage for 2 days in control medium, in 2i
(medium supplemented with PD0325901 and Chir99021), or medium supplemented
with PD0325901. Embryos were immunostained using antibodies raised against
Oct4 (red), Nanog (green) and Gata4 (blue) and nuclei were counterstained with
DAPI. (D) Bar chart showing cell numbers of epiblast (green) and
hypoblast (blue) of embryos cultured in the conditions shown in C. Bars
indicate the mean ± s.d.

Effect of 3i/2i applied at the blastocyst stage

The absence of hypoblast in inhibitor-treated embryos could be due to
selective ablation of this lineage. However, the maintenance of total ICM
number in embryos cultured for 2 days in 2i compared with controls
(Fig. 1C,D) suggests the
alternative explanation that cells assigned to the ICM might have all been
directed to the Nanog-positive lineage in preference to the Gata4-positive
domain. In an attempt to distinguish between these possibilities, embryos were
collected at the expanded blastocyst stage at ∼3.75 dpc (E3.75), when the
hypoblast is thought to be already determined
(Chazaud et al., 2006), and
cultured with or without inhibitors for 2 days. Numerous Gata4-positive cells
were present at the end of this period in 3i, 2i and control culture
conditions (Fig. 2A). This
indicates that the inhibitors are not acutely toxic to hypoblast cells.
Therefore, the absence of Gata4-positive cells in embryos treated from the
8-cell stage might be a result of preferential direction of the ICM cells into
epiblast, rather than selective destruction of hypoblast.

During normal development, as embryos approach implantation, Nanog is
transcriptionally downregulated in the epiblast until it becomes undetectable
(Chambers et al., 2003;
Hart et al., 2004). Freshly
isolated peri-implantation stage embryos retain a few Nanog-positive cells,
detectable by immunostaining (Fig.
2A). Embryos cultured from the blastocyst stage in control
serum-free medium lose expression of Nanog completely, although Oct4
expression persists in a subset of ICM cells that is distinct from the
Gata4-positive domain (Fig.
2A). By contrast, embryos cultured in serum-free medium with
inhibitors (3i or 2i) maintained high levels of Nanog protein in the epiblast
(Fig. 2A). Strikingly,
expression of Oct4 was not restricted to the Nanog-positive domain, but was
also maintained in the hypoblast in blastocysts
(Fig. 2A). This profile of
Nanog and Oct4 double-positive epiblast and Oct4-positive hypoblast is
maintained in embryos in diapause
(Batlle-Morera et al., 2008),
the stage of arrested development from which ES cells are most readily derived
(Batlle-Morera et al., 2008;
Brook and Gardner, 1997;
Kawase et al., 1994). Since
Oct4 expression is progressively lost from the hypoblast during embryo
maturation in vivo and in control culture conditions
(Fig. 2A), the persistence of
Oct4 in hypoblast during diapause and in 3i/2i blastocyst culture might
reflect a block in developmental progression that maintains the embryo at the
optimal stage for ES cell derivation.

Inhibition of hypoblast formation in early embryos is not
reversible

Once the hypoblast has been microsurgically removed from epiblasts of
peri-implantation mouse embryos it is not regenerated
(Gardner, 1985). Moreover,
hypoblast is not produced from mouse epiblasts mechanically isolated from
diapause blastocysts (Batlle-Morera et al.,
2008; Brook and Gardner,
1997). To investigate whether hypoblast can be regenerated from
epiblasts developed in inhibitors, we incubated embryos from the 8-cell stage
in 2i for 2 days, then transferred them to control medium for a further 2 days
prior to fixing and immunostaining. Only one of seven embryos cultured in this
way exhibited any Gata4-positive cells. One of six embryos cultured in 2i for
the entire 4 days also exhibited a few Gata4-positive cells. By contrast, all
six embryos cultured for 4 days in control medium possessed large numbers of
Gata4-positive cells (Fig.
2B,C; P<0.0001). The irreversible loss of hypoblast
formation imposed by the inhibitors is consistent with commitment to the
epiblast, indicating that ICM cells in treated embryos do not arrest at an
early ICM stage.

Formation of functional epiblast in embryos cultured in ground state
conditions. (A) Confocal images of mouse embryos freshly isolated
at 4.5 dpc (E4.5), embryos cultured for 2 days from E3.75 in control medium,
in 3i or in 2i. Embryos were immunostained using antibodies against Oct4
(red), Nanog (green) and Gata4 (blue). (B) Confocal images of embryos
grown from the 8-cell stage (E2.5) for 4 days in 2i, for 2 days in 2i then a
further 2 days in control medium, or in control medium for 4 days. Embryos
were immunostained as in A. (C) Bar chart showing cell numbers of
epiblast (Oct4 positive, Gata4 negative, green) and hypoblast (Gata4 positive,
blue) of embryos cultured in the conditions shown in B. Bars indicate the mean±
s.d. (D) Confocal images of embryos freshly isolated at E4.5,
embryos cultured from the 8-cell stage (E2.5) for 3 days in 2i, or from E3.5
for 2 days in 2i. Embryos were immunostained using antibodies against Eed
(green) and Gata4 (red). Scale bars: 20 μm. (E) Mice generated from
injection of isolated epiblast cells from embryos grown for 3 days in 2i.
Donor cells were from 129/Sv embryos (agouti coat colour); host blastocysts
were of the C57BL/6/O1a strain (black coat colour). (F) One of the
female chimaeras and her C57BL/6/O1a mate with their offspring comprising
seven agouti pups, demonstrating transmission through the germline of the
donor cell genotype, and one black offspring produced from the germ cells of
the host embryo. (G) Summary of blastocyst injection experiments to
test the developmental capacity of epiblast cells from embryos cultured in
ground state culture conditions. Asterisk indicates that nine mice were tested
for germline transmission.

ICM cells develop into functional epiblast in 3i/2i

We investigated the epigenetic status of ICM cells generated in 2i by
examining the X chromosomes in female embryos. The Polycomb group protein Eed
marks the inactive paternal X chromosome in early embryos
(Mak et al., 2004;
Okamoto et al., 2004). In
female blastocysts at E3.5, all cells carry an inactive X chromosome.
Reactivation of the silent X chromosome occurs exclusively in the epiblast
between E3.5 and E4.5 (Silva et al.,
2009). Accordingly, a prominent nuclear focus of immunostaining
corresponding to enrichment of Eed on the inactive X could be detected in the
hypoblast and trophectoderm cells of female embryos at E4.5, but was absent in
the epiblast (Fig. 2D). In
female embryos cultured in 2i, the ICM cells lacked Eed foci, implying that X
reactivation had occurred. The erasure of X chromosome silencing substantiates
the argument that cells transit from ICM to epiblast state in the presence of
2i.

To confirm the development of functional epiblast in embryos cultured in 2i
from the 8-cell stage, we disaggregated the ICMs and injected them into
blastocysts. From 32 blastocysts injected, 11 coat colour chimaeras were
obtained. Seven of these subsequently exhibited germline transmission of the
injected cells (Fig. 2E-G).
These results establish that the inhibitor treatment does not compromise
epiblast identity or potency. This suggests that the inhibitors hold
naïve epiblast cells in the ground state, from which they can readily
resume normal development.

Culture from the zygote stage in 2i reduces the number of
trophectoderm cells

To determine whether culture in 2i affects development of the
trophectoderm, we cultured embryos with or without inhibitors from the zygote
stage for 5 days before fixation and immunostaining. Treated embryos formed
expanded blastocysts that hatched from the zona pellucida, properties that are
dependent on functional trophectoderm (Fig.
3A). Immunostaining for Cdx2 and Eomes showed that these
trophoblast markers were expressed in the outside cells of embryos cultured in
inhibitors (Fig. 3B; data not
shown). These results indicate that, unlike hypoblast, formation of
trophectoderm is not dependent upon Erk signalling. However, embryos cultured
in 2i or in PD0325901 alone developed significantly fewer trophectoderm cells
than the controls (Fig. 3C;
P<0.0001), consistent with reports that trophoblast proliferation
is stimulated by Fgf4 (Nichols et al.,
1998; Tanaka et al.,
1998). As with culture of embryos from the 8-cell stage,
Nanog-positive epiblast cells increased in number at the expense of
Gata4-positive hypoblast in ICMs formed in the presence of 2i or PD0325901
(Fig. 3;
P<0.0001).

Effect of LIF on epiblast cells in embryos cultured in 2i and
explanted. (A) Confocal images of mouse embryos cultured from the
8-cell stage for 2 days in 2i or 2i+LIF. Embryos were immunostained using
antibodies against Oct4 (red), Nanog (green) and Gata4 (blue). (B) Bar
chart showing cell numbers of epiblast (green) and hypoblast (blue) of embryos
cultured in the conditions shown in A. Bars indicate the mean ± s.d.
(C) (Left) Bright-field image of a single epiblast cell immediately
after isolation from an embryo cultured for 3 days in 2i+LIF, plated into one
well of a 96-well plate. (Right) Bright-field image of a colony produced from
the same cell after growth in 2i+LIF for 8 days. Scale bars: 20 μm.
(D) Summary of colonies produced from single epiblast cells plated into
2i or 2i+LIF. (E) Confocal images of a colony grown from a single
epiblast cell plated into 2i+LIF for 8 days, as shown in C. Colonies were
immunostained using antibodies against Oct4 (red) and Nanog (green) and nuclei
were counterstained with DAPI. (F) Bar chart showing the number of ES
cell clones produced per embryo from single ICM cells isolated from freshly
flushed F1 embryos at E4.5.

Addition of LIF does not enhance the response of embryos to 2i

Although derivation and culture of ES cells in 3i/2i medium does not
require the addition of any cytokines, clonogenic efficiency is improved by
supplementation with LIF (Ying et al.,
2008). However, we found that addition of LIF to embryos cultured
in 2i did not enhance expansion of the ICM
(Fig. 4A,B). This might be
because endogenous LIF is sufficient. In the embryo, the components of LIF
signalling are present by the blastocyst stage, with LIF expressed
specifically by the trophectoderm, and the receptor components predominating
in the ICM (Nichols et al.,
1996).

Clonal propagation of ES cells from dissociated epiblast

ES cells have been derived from single epiblast cells, but at low
efficiency (Brook and Gardner,
1997). This has provoked discussion as to whether epiblast cells
are equipotent or whether only a minority have the capacity to become ES cells
(Gardner and Brook, 1997). We
observed that the derivation of ES cells from embryos cultured in 3i/2i from
the 8-cell stage was very efficient (Ying
et al., 2008). We therefore used this system to address the
ability of the individual epiblast cells to form ES cells. We used
immunosurgery to isolate ICMs from strain 129 embryos cultured for 3 days in
2i from the 8-cell stage. We dissociated the ICMs and deposited single cells
into individual wells of 96-well plates in 2i or 2i plus LIF. Of individual
cells plated in 2i alone, two out of 33 (6%) gave rise to undifferentiated ES
cell colonies. By contrast, 25 out of 46 cells (54%) plated in 2i plus LIF
yielded ES cell colonies (Fig.
4C,D). Addition of LIF to 3i has previously been shown to enhance
clonogenicity of ES cells, presumably by activation of the Stat3 pathway
(Ying et al., 2008). The
colonies produced in 2i alone were morphologically indistinguishable from
those derived in 2i plus LIF. Several colonies were stained for Oct4 and Nanog
and were found to exhibit uniformly high levels of both pluripotency markers
(Fig. 4E). The remaining
colonies were expanded as ES cell lines. Two of these were injected into
blastocysts and gave rise to chimaeras that subsequently transmitted the ES
cell genome through the germline (data not shown).

Finally, to exclude the possibility that prior culture of embryos in 2i
induces some epigenetic adaptation, we isolated and dissociated the ICMs from
freshly harvested E4.5 blastocysts and plated single cells or couplets into
individual wells of 96-well plates containing 2i plus LIF. All ICMs produced
colonies. The mean number of undifferentiated colonies per embryo scored after
8 days in culture was 5.75 (n=12), ranging from two to 12 colonies
(Fig. 4F). These data indicate
that naïve epiblast cells in the murine embryo can transit directly to
self-renewing ES cell status. Furthermore, approximately half of the cells
plated from E4.5 ICMs will be committed hypoblast, and given that the cloning
efficiency of established ES cells is less than 50%
(Ying et al., 2008), the
frequency of ES cell colony formation suggests that all epiblast cells are
likely to have this property.

ICMs of embryos developed to the blastocyst stage in the presence of
FGF/Erk signalling inhibition exhibit uniformly high levels of Nanog or Oct4
(Fig. 1;
Fig. 2A,B). They entirely lack
overlying hypoblast, yet have similar or even increased total ICM cell numbers
compared with embryos cultured without inhibitors or freshly isolated
peri-implantation embryos. In embryos cultured in the Gsk3 inhibitor alone,
hypoblast formation is not overtly impaired
(Fig. 1A,B). This result
appears at variance with a recent report in which ES cells were efficiently
derived from embryos of recalcitrant strains of mice using the Gsk3 inhibitor
BIO, and ICM outgrowths specifically exhibited reduced hypoblast proliferation
(Umehara et al., 2007).
However, BIO is less selective for Gsk3 than Chir99021 and is likely to
inhibit various other kinases including Cdks, FGF receptor and, possibly, Erk
(Zhen et al., 2007).
Furthermore, in the Umehara study, the outgrowths were maintained on murine
embryonic fibroblast feeders in medium supplemented with serum.

The mechanism underlying the absence of hypoblast in embryos cultured in 2i
is likely to be a fate change rather than specific ablation. Embryos exposed
to the inhibitors at the expanded blastocyst stage of development, when the
hypoblast is already specified, develop numerous Gata4-positive cells
(Fig. 2A). Furthermore, the
diversion of ICM cells to an epiblast fate by inhibition of FGF/Erk signalling
seems irreversible, as further culture of embryos following removal of
inhibitors does not restore the hypoblast
(Fig. 2B,C). This suggests that
the ICM cells in 2i undergo normal developmental progression and lose the
capacity to form hypoblast coincident with acquiring pluripotent epiblast
identity. Transition to epiblast is further indicated by erasure of epigenetic
silencing of the inactive paternal X chromosome in female embryos cultured in
2i (Fig. 2D). The presence of
two active X chromosomes is a hallmark of the pluripotent ground state of ES
cells that is not shared by epiblast stem cells (EpiSCs), extra-embryonic
trophectoderm stem (TS) or extra-embryonic endoderm (XEN) cell lines
(Guo et al., 2009). Female
human ES cell lines vary in the activation status of their paternal X
chromosome; they are apparently subject to dynamic epigenetic reprogramming ex
vivo that is not necessarily reflective of their pluripotent status
(Silva, S. S. et al., 2008).
Neither human ES cells nor EpiSCs exhibit X chromosome reactivation when
cultured in 3i/2i, and ultimately fail to survive
(Nichols and Smith, 2009). We
show in this study that epiblast cells from embryos cultured in 2i are able to
contribute normally to embryonic development and form viable chimaeras and
functional germ cells when injected into host blastocysts
(Fig. 2E-G). These results
demonstrate that functional epiblast can develop when FGF/Erk and Gsk3
signalling are inhibited, and that neither the development nor potency of
murine epiblast requires interaction with hypoblast.

In contrast to the requirement for development of hypoblast, specification
of the trophoblast lineage does not appear to require activation of the Erk
pathway. Embryos cultured in the presence of 2i from the single-cell stage
cavitated and hatched from the zona pelucida, demonstrating the formation of
functional trophectoderm. Moreover, the cells of this outer epithelium
expressed both Cdx2 and Eomes, two definitive markers of trophectoderm
(Fig. 3B; data not shown).
Nonetheless, expansion of the trophectoderm is affected by inhibition of Erk
activity; embryos cultured in 2i or in PD0352901 alone exhibited a decrease in
trophectoderm cell numbers compared with controls
(Fig. 3). Expression of FGF
receptors has been demonstrated in trophectoderm and its derivative
extra-embryonic ectoderm (Arman et al.,
1998; Holdener et al.,
1994; Rappolee et al.,
1994), and a requirement for FGF signalling has previously been
implicated in proliferation of the diploid population of cells that reside in
the polar trophectoderm (Nichols et al.,
1998; Tanaka et al.,
1998). Thus, Erk signalling appears to be dispensable for
trophectoderm specification and differentiation in the early embryo, but is
likely to be required for maintenance of a proliferating diploid
population.

We have proposed that naïve pluripotency might be a basal mammalian
cell state that is intrinsically self-maintaining if shielded effectively from
inductive differentiation stimuli (Ying et
al., 2008). The present findings indicate that this state is
shared between pre-implantation epiblast and ES cells. The intrinsic
self-replication exhibited by mouse ES cells is thus not an adaptation to
culture, but directly represents autonomous expansion of early epiblast. In
the unperturbed embryo, self-renewal is short-lived owing to the inductive
action of Fgf4 and other extrinsic Erk stimuli, but this feed-forward stimulus
can be arrested in diapause, during which the epiblast can remain in a
naïve state for weeks. ES cell lines can most readily be derived from
diapause blastocysts (Kawase et al.,
1994). Upon explant culture, the epiblast will rapidly lose
pluripotency and differentiate under the influence of Erk signalling
(Buehr et al., 2003;
Buehr and Smith, 2003).
Disrupting this signal with 3i/2i prevents this progression and releases ES
cells. This has been exploited to enable efficient derivation of ES cells from
embryos of recalcitrant CBA and MF1 mouse strains
(Ying et al., 2008), NOD mice
(Nichols et al., 2009) and
rats (Buehr et al., 2008;
Li et al., 2008). We conclude
that in rodents, naïve epiblast and ES cells are the same ground state
entity, differing only by their environment. It will be important to determine
whether this principle extends to other mammals, in particular primates, from
which authentic ES cells have yet to be described.

Footnotes

We thank Keith Savill and staff for animal husbandry, and Brian Hendrich
and Jason Wray for helpful comments. This research was funded by the
Medical Research Council
(G9806702ID84670) and the Wellcome
Trust (073607).
A.S. is a Medical Research Council Professor. Deposited in PMC for release
after 6 months.

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